The goal of this research is to understand the mechanism of biosynthesis of bacterial peptide siderophores. We use as a paradigm the peptide siderophore anguibactin that is produced by the pathogenic bacterium Vibrio anguillarum. Anguibactin is an important component of the pJM1 plasmid-mediated iron uptake system that is essential for virulence of these pathogenic vibrios. Genetic and physiological analysis led us to the identification and cloning of genes encoded on the pJM1 plasmid that play an essential role in anguibactin biosynthesis. DNA sequence and protein analysis revealed that these genes encode polypeptides that possess domains found in nonribosomal peptide synthetases (NRPSs), originally identified as components of the biosynthetic machinery for the synthesis of antibiotics in gram-positive bacteria. These proteins have been named AngB, AngM, AngN, and AngR and possess modules that could be involved in one or more of the following reactions during the biosynthesis of anguibactin: peptidyl carrier protein (PCP), involved in thioester formation; condensation (C), intervening in peptide bond formation; cyclization (Cy), involved in both condensation and heterocycle formation, and adenylation (A), which is responsible for substrate activation. AngB is an isochorismate lyase that also operates as an aryl carrier (ArCP) protein during siderophore assembly. Other proteins encoded by plasmid-mediated genes, such as AngH and AngU, possess enzymatic activity for the synthesis of histamine from histidine, and for the further oxidation of this compound to hydroxy-histamine, which is a basic building block of anguibactin. Our present efforts are thus directed to elucidate the role of the specific modules of the NRPSs in siderophore biosynthesis. The specific aims to achieve this goal are: 1. Dissection of the mechanisms of assembly line enzymology of anguibactin biosynthesis. We have purified these NRPSs proteins and have obtained antibodies which will be used in the assessment of the role of these polypeptides by using in vitro synthesis reactions including swapping of equivalent NRPS modules intervening in siderophore biosynthesis in V. anguillarum, Vibrio cholerae, and other pathogens. 2. Mutational analysis of the NRPSs genes that will include random mutagenesis and site-directed mutagenesis of the specific modules. We will also purify selected mutant proteins to be used in in vitro synthesis reactions to identify single steps during anguibactin assembly. 3. Identification of chromosomal-encoded proteins intervening in anguibactin biosynthesis. We will use a combination of transposon-directed cloning, genetic, immunological and biochemical approaches to characterize these genes. The combination of the in vivo genetic and the in vitro biochemical approaches will likely lead to the dissection of the mechanisms of siderophore biosynthesis and, in turn, to the exploration of new avenues to understand this contribution to bacterial virulence.